Laboratory diagnostics

Platelet counts are usually included in every complete blood count. They can, however, also be requested individually. Additionally, you should also always prepare a blood smear directly before or after platelet analysis. This blood smear can be used to look for aggregates when platelet counts are low or to confirm thrombocytopenia. To evaluate the blood smear, it is air-dried and then stained. You can, for example, use Wright-Giemsa or Diff-Quik for staining (Figures 2a and 2b).
For further evaluation, a detailed clinical history (e.g. contact with viral, bacterial, parasitic infectious agents? Fever? Gastrointestinal signs? Respiratory signs?) and a thorough examination of the patient are necessary.

Microscopic examination of the platelets is first carried out at 100x magnification (10x objective). The feathered edge is examined for platelet aggregates. Platelets are then counted in the monolayer at 1000x magnification (100x objective). In order to calculate the approximate platelet count, the average of 10 visual fields is multiplied by 15 – 20. Any aggregates in the feathered edge must also be taken into account. This blood smear can be used to look for aggregates (Fig. 3) when platelet counts are low or to confirm thrombocytopenia.  For example: In the monolayer, 98 platelets are counted in 10 visual fields. The average is 9.8 platelets per visual field. The number is rounded up to 10 and then multiplied by 15.
Thus, the estimated platelet count of the horse is 150 G/l. Rough estimates of any aggregates must be added to this figure.

If thrombocytopenia is confirmed by the blood smear, further examinations are recommended depending on the clinical history and the suspected diagnosis, e.g.

• • PCR tests: e.g. Babesia, Anaplasma
  • coagulation tests: PT, aPTT, thrombin time, fibrinogen
  • serological tests: e.g. equine infectious anaemia (EIA), equine herpesvirus (EHV)
  • haematology: antiplatelet antibodies
  • bone marrow biopsy and cytology, if necessary

Thrombocytopenia

In most cases, thrombocytopenia is an incidental finding in horses. Thrombocytopenia is a decrease in platelet numbers below the species-specific norm.⁹ Thrombocytopenia usually indicates a pathological process which can lead to coagulation disorders when platelet count is significantly reduced.¹⁰ There is often more than one cause for thrombocytopenia.¹²
A distinction must be made between pseudothrombocytopaenia and true thrombocytopenia. Pseudothrombocytopenia occurs if not all the platelets present are counted in the platelet count. Manual microscopic examination is therefore always recommended as a first control mechanism in thrombocytopenia.¹⁰
Reasons for true thrombocytopaenia are reduced production in the bone marrow, increased consumption, destruction of platelets or increased sequestration (Table 1).^{10, 11}

Table 1: Summary of causes of thrombocytopenia ^{10, 11}

Cause
Thrombocytopenia	pseudothrombocytopenia	limitations of measurement platelet aggregation due to platelet activation macroplatelets EDTA-induced (very rare)
	reduced production in the bone marrow	bone marrow aplasia megakaryocyte leukaemia myelofibrosis myelonecrosis metastases medication toxins radiation genetic
	increased consumption	severe bleeding disseminated intravascular coagulation (DIC) haemolytic uraemic syndrome (HUS) neoplasia
	destruction of platelets	immune-mediated destruction primary immune-mediated thrombocytopenia secondary infectious diseases neoplasia medication alloimmune thrombocytopenia Evans syndrome non-immune-mediated destruction infectious diseases viruses medication heparin-induced toxins snakebite
	sequestration	splenomegaly severe hypothermia endotoxaemia

 

Clinical signs of thrombocytopenia
In most horses, thrombocytopenia occurs secondary to a variety of underlying diseases.¹²
The clinical picture usually corresponds to the causative disease. In general, signs are rare in low to moderate thrombocytopenia (90 – 30 G/l) as long as secondary haemostasis is not impaired.
Severe thrombocytopenia (< 30 G/l) leads to changes in primary haemostasis.^{13, 14} Patients show petechiae and ecchymoses, which are mainly seen on the mucous membranes and the sclerae. Increased epistaxis has also been described. There is prolonged bleeding after blood sampling.

Thrombocytosis

Thrombocytosis is defined as an increase in the number of platelets circulating in the peripheral blood above the species-specific reference range. As with thrombocytopenia, a distinction can be made between pseudothrombocytosis and true thrombocytosis (Table 2). Pseudothrombocytosis occurs when other cells are counted as platelets in the platelet count. This can include small erythrocytes or lysed erythrocytes. The evaluation of scatterplots and blood smears helps here.

Mild, physiological thrombocytosis often occurs during and after exercise or agitation (e.g. stress during blood collection). This is caused by splenic contractions which release stored platelets into the circulation.^{18, 19}
True thrombocytosis can be classified into primary and secondary thrombocytosis. In most cases, platelet count increase in secondary thrombocytosis is only mild to moderate (300 – 500 G/l), whereas it is high in primary thrombocytosis. Furthermore, primary thrombocytosis is more frequently associated with clinical bleeding and thrombosis.^{20, 21, 22}
Secondary thrombocytosis mostly occurs in inflammation and infections.²⁴

Table 2: Summary of causes of thrombocytosis ^{11, 22, 23}

Cause
Thrombocytopenia	pseudothrombocytosis	limitations of measurement lysed erythrocytes (“ghost cells”)
	redistribution of platelets	exercise catecholamines
	increased production in the bone marrow	inflammation infection immune-mediated surgical intervention trauma non-haematopoietic neoplasms iron deficiency recovery from thrombocytopenia (rebound) splenectomy (post) haemorrhage
	neoplasia	primary (essential) thrombocythemia acute megakaryoblastic leukaemia (as of 2021 not described in horses) chronic myeloproliferative neoplasms

Conclusion

Platelet counts are part of almost every blood count and may provide an indication of an underlying disease. In any case, their plausibility must first be checked manually under the microscope. The cause of thrombocytopenia or thrombocytosis must then be found based on a good medical history and clinical examination.

Dr. med. vet. Annemarie E. Baur-Kaufhold

The selection of allergens should be based on the clinical picture.
They are selected for the ASIT:

1. 1. allergens with a positive reaction in the allergy test (regardless of the intensity of the reaction)
   2. that correlate with the history and clinical signs
   3. that are present in the animal‘s environment
   4. the number of allergens to include is limited, so in cross-reacting allergens, the representative allergen of the group or a mixture of cross-reacting allergens is used

At the beginning of therapy, medical treatment is usually necessary to control respiratory signs. If medical treatment is used, a treatment at minimal dosage that alleviate the clinical signs is advisable to be able to adapt the therapy plan.
The administration of subcutaneous immunotherapy begins at weekly intervals and progresses to monthly injections. Initially the therapy begins with a low dose of allergens extract which is increased until reaching the maintenance dose. The goal is to develop tolerance to injected allergens so that clinical signs triggered by an overreaction of the immune system disappear. ASIT is the only therapy that intervenes in the primary origin of the disease. In veterinary medicine, if the response to this therapy is good, it is recommended to continue it for life since allergies are not curable, and experience has shown that after discontinuation of treatment, the clinical picture usually reappears after 1 to 2 years. The veterinarian should monitor the animal regularly, as adjustments in the injection interval and/or the dose may be necessary.

For the owner, the success of the therapy is crucial. Our studies in horses with allergic respiratory diseases show a clear improvement in the clinical picture in more than 80% of the patients included. If therapy was started within the first two years after the first respiratory signs appeared, the chances of success increased to 86%. ASIT is an extremely promising therapy, especially for horses with respiratory problems, which often become impossible to ride due to this disease.

Clinical picture	ASIT success
Respiratory disease	80%
ASIT within 2 years of respiratory disease	86%

 

In addition, it should be noted that all allergic events have a genetic component, i.e. affected animals should be excluded from breeding, which is particularly important for breeding stallions with a high number of offspring.

Dr. Regina Wagner

One author also found out that horses which had a proven history of summer eczema on the continent still exhibited significant sensitisation of their basophilic granulocytes to Culicoides after 15 asymptomatic years in Iceland. Even after such a long time, these animals had not lost their basic predisposition to develop summer eczema again when exposed to an appropriate allergen. In another study, Icelandic horses were imported from Iceland to Sweden. Horses which arrived in winter were twice as likely to develop IBH as those that arrived in summer. In Iceland, the animals have a high IgE titre due to worm infestation. Through endoparasite management after importation, it decreases, remains low in the healthy animal and increases again in animals suffering from eczema. When horses are imported in the summer, IgE against Culicoides competes with the antibodies produced due to the presence of endoparasites. During the winter IgE associated with endoparasites have decreased and there is no interference with IgE against Culicioides.

It was also found that IgE antibodies can be transferred from mother to foal via the colostrum. Both serum and cell-bound IgE could not be detected in unborn foals, but there was a clear peak 2 – 5 days after colostrum intake. These “maternal” antibodies are detectable in young animals during the first 4 months of life.
According to this study, endogenous IgE production begins at the earliest at an age of 9 – 11 months.

The first signs are usually seen at a young age (2 – 4 years). Generally, the clinical picture is strictly seasonal, highly pruritic and may be accompanied by papules or wheals.

All other efflorescences occur secondarily: alopecia, scales, crusts, excoriation, hyperpigmentation and lichenification. A dorsal and/or ventral distribution pattern is frequently seen. If the mane or the tail are affected, all that will typically remain is only a crusty hairless crest and a so-called “rattail”. Secondary bacterial infections of the damaged and traumatised skin are common and aggravate the itching even further.

The main differential diagnoses for insect bite hypersensitivity are atopic dermatitis (allergy to environmental allergens such as pollen, house dust and storage mites as well as moulds), food allergy, secondary bacterial infections and ectoparasites (biting lice/lice, Chorioptes and Psoroptes mites). Clearly, many animals do not only suffer from an allergic reaction to insects, but also to other allergens. An important indication in these cases is that the signs do not completely disappear in winter.

Diagnosis of insect bite hypersensitivity is based on the medical history (a single horse affected, seasonality) and the clinical picture (distribution pattern of pruritus and efflorescences). Skin scrapings should always be routinely examined to exclude the presence of ectoparasites. Cytology is also recommended for the diagnosis of secondary infections. In many cases, histopathology confirms the suspected diagnosis of an allergy, but does not provide any information on the type of allergen.

An allergy test can determine which insect species or which other seasonal allergens (e.g. pollen) are involved in the present disease.

Serological allergy tests offer a good opportunity to determine the causative allergen, but, as with other allergies, do not provide a diagnosis. The positive result is correlated with the medical history as well as the clinical picture and used to avoid allergens or to formulate ASIT (allergen-specific immunotherapy). Basically, it is necessary to distinguish between a skin allergy test (intradermal test), serological tests in which allergen-specific IgE are detected (ELISA), and cell stimulation tests. Very intensive research is carried out in order to establish allergy tests with recombinant allergens. In April 2021, for example, a study was published in which a microarray with Culicoides recombinant allergens was developed. There were 27 recombinant allergens of which 9 were major allergens for IBH with 7 of them being positive in more than 70% of IBH-affected horses.

Therapeutically, management is of great importance. Horses must be kept away from insects as much as possible. Various measures can be considered: keeping horses in a stable during the day and on the paddock at night, fine insect netting, fan in the stable, removal of standing water near the stable (old car tyres, cans …), eczema blankets, insect control with effective fly sprays (permethrin – caution: stable cats!, pyriproxyfen or icaridin).

Shampoo therapy or simply hosing down with cold water should also not be underestimated in order to reduce the percutaneous allergen load. Additionally, possible secondary bacterial infections are treated using an antibacterial agent (e.g. chlorhexidine 2 – 4%). The use of topical glucocorticoids is certainly justified in localised itchy areas. However, it is always important to bear in mind the withdrawal time (four weeks for topical preparations) in case an allergy test needs to be carried out!

Allergen-specific immunotherapy (ASIT, hyposensitisation) is the only treatment that provides causal intervention in the disease. It is used for insect bite hypersensitivity, urticaria caused by environmental allergens, atopic dermatitis, equine asthma or headshaking. In case of seasonal diseases, treatment should be started at the end of the season, i.e. in autumn for animals suffering from summer eczema. Therapy must be carried out for at least 12 months in order to be able to accurately determine its efficiency. If it is successful, it should be carried out for the rest of the horse’s life. The allergens included must correlate with the medical history and the clinical picture and should be selected specifically for the patient. The success rate of ASIT ranges from 60% to 70%. If the test result is positive (intradermal or blood allergy test) and correlates with the medical history and the clinical picture, this form of therapy is a treatment option which is rarely accompanied by side effects and which is cost-effective (weight-independent costs (!)). After the initial treatment phase, only occasional applications are required in the further course of the disease and this implies, in addition to cost efficiency, less effort for the owner (the maintenance dose is usually a subcutaneous injection every 4 weeks). This type of treatment can also be carried out on sport horses, as these animals often return to a good performance level and yet are not treated in a doping-relevant way.

New promising treatments, such as an active vaccine against IL-5 or IL-31, have been published but are not yet ready for the market.

In summary, it should be noted once again that allergies are very common in horses, the disease is not curable, but various diagnostic and therapeutic options allow for good management of the disease as long as the owner is properly informed and willing to cooperate.

Dr. Regina Wagner

Symptoms

11 years ago, an unusual outbreak of fever and enteric symptoms in 2-4 year old racehorses were seen in Japan. Only 10% of these horses showed enteric signs out of the 132/600 (22%) horses that became sick. The same racing venue experienced an additional outbreak with similar signs three years later. Outbreaks have since been observed throughout Europe and the USA.

Common symptoms, in clinical affected horses are fever, anorexia, lethargy and/or enteric symptoms (in about 10% the cases). The lack of enteric symptoms in adult horses, such as colic and/or changes in faecal character, may relate to the intestinal section affected by the virus. ECoV has been shown to cause enteritis in both foals and adult horses. While enteritis is consistently associated with diarrhoea in foals, it may not affect the faecal character of infected adult horses, which might only show colic symptoms, if any enteric symptoms at all.

Most infected adult horses recover with minimal or no medicinal treatment within 2-4 days, while some may require intensive care to resolve leukopenia, systemic inflammation and metabolic disturbances. Morbidity rates have been reported to range between 17-57%, with mortality rate being very low.

Complications are rare and have been associated with disruption of the gastro-intestinal mucosal barrier leading to endotoxaemia, septicaemia and hyper-ammonemia-associated encephalopathy.

Of interest is the observation that clinical expression of ECoV infection is age-dependent with foals rarely developing clinical disease. Given the lack of documented outbreaks at large breeding farms, it is possible that circulating virus amongst foals and breeding stock confers protection against clinical disease. A recent study supports this, as they found higher seroprevalence to ECoV in healthy breeding animals, compared to non-breeding horses.

The lack of gastrointestinal symptoms often misleads the equine practitioner into ruling out an enteric pathogen. Haematological finding, such as leukopenia due to neutropenia and/or lymphopenia, although not specific for ECoV, should direct the diagnostic workup towards a viral disease.

Diagnosing ECoV

RT-PCR is used to detect ECoV in faeces, and has proven to be more sensitive and specific than other test methods, such as electron microscopy and antigen capture ELISAs, RT-PCR test has a quicker turnaround time and is  more cost-effective.

It appears that longer duration and higher peaks of viral shedding are observed in clinically versus non-clinically infected horses, although both groups may contribute to environmental contamination and viral transmission.

While several factors, such as viral strain, age of patient and co-morbidity influence the outcome of infection, a recent study was able to associate ECoV viral load measured by RT-qPCR with mortality, as seen by other corona-viruses, such as Feline Coronavirus (FCoV) and the human SARS-CoV-2.

Due to the rapid autolysis of the gastro-intestinal tract, it is relevant to have a necropsy performed rapidly or have representative samples collected and frozen for ECoV detection and placed in formalin for pathological evaluation. ECoV can be diagnosed post-mortem by RT-PCR on faeces or small intestinal biopsies, submitted without fixation.

Prophylaxis

There are no vaccines against ECoV available yet, and specific preventive measurements are scarce. Due to the close genetic homology of ECoV with BCoV, serological responses to BCoV vaccines have recently been investigated, showing measurable antibodies against BCoV. These are not recommended to use at this time, due to the lack of efficacy data.

The cornerstone of ECoV prevention is strict biosecurity measurements aimed at reducing the risk of introducing and disseminating ECoV on the premises.

Conclusion

It is important to be thorough, when working-up horses presenting with fever, anorexia and lethargy, with or without concurrent enteric symptoms (mostly colic signs in adults and diarrhoea in foals). And important to isolate such horses until ECoV, as well as other potential infectious pathogens (e.g. Influenza A virus, Equine Herpesvirus 1 and 4, Streptococcus equi equi), have been ruled in or out by PCR.
It may be wise to test the population to uncover subclinically infected individuals as well, as they may shed the virus too.

ECoV PCR-positive horses should be isolated and stable or herd mates closely monitored until the outcome of past-exposure has been determined.

Outbreaks of ECoV are generally short lasting, especially when strict biosecurity measures have been followed, and quarantine can routinely be lifted 2-3 weeks following the resolution of clinical symptoms in the last affected horse.
Common disinfectants inactivate ECoV, but it is not known how long ECoV remains infectious in the environment. SARS-CoV-2 has been shown to persist up to 2 days in wastewater and dechlorinated tap water, 3 days in faeces and 17 days in urine at room temperature. The survival of the virus is even longer at lower temperatures.

In conclusion, equine practitioners should take advantage of the knowledge gained over the past decade in the field of ECoV in adult horses and foals. Although further investigations are needed, we have more insights to the disease, its clinical picture, diagnostic tools available and treatment modalities in place, in order to have a successful outcome of horses infected with ECoV.

Charlotte Hoffmann-Timmol, DVM und Dr. Svenja Möller